Although digital measurement methods such as single-molecule array (Simoa) or digital enzyme-linked immunosorbent assay (dELISA) have achieved breakthroughs in sensitivity of measuring extremely low levels of proteins in various biological samples, the complexity of their equipment has restricted their wide application in clinical diagnosis. To overcome these limitations, we developed the Digital Immunoassay via Single-molecule Counting and Accurate Numerical analysis (DISCAN). This method combines an immunosandwich assay with rolling circle amplification (RCA) to enable direct digital quantification of single-molecule fluorescence signals on beads, eliminating the need for Poisson distribution-based confinement in microwells. This platform has achieved an attomolar detection limit, exhibiting over a 1000-fold higher sensitivity than conventional ELISA and a 6-fold improvement over single-molecule array (Simoa). Using DISCAN, we measured plasma p-tau217 levels in patients with Alzheimer's disease (AD) and age-matched healthy controls. Elevated p-tau217 levels were observed in the AD group, supporting DISCAN’s clinical applicability. With its markedly enhanced sensitivity and convenient design, DISCAN provides a new approach for low-abundance protein detection, particularly holding promise as a pivotal tool for the early diagnosis of Alzheimer's disease.